Immune complexes are formed in the blood stream between antigen and antibody as part of the normal immune defense mechanism against pathogens and other foreign substances. These immune complexes allow elimination or neutralization of the foreign antigens and protection of the host. Under some circumstances, however, these immune complexes may themselves cause injury to the host. These circulating immune complexes are relevant to a large number of diseases, especially autoimmune diseases such as rheumatoid arthritis, systemic lupus erythemathosis (SLE), and the like; cancers such as leukemia and ovarian cancer; and a large number of infectious diseases. Particularly in autoimmune diseases, it is useful to detect circulating immune complexes in diagnosing the disease and in monitoring its treatment. See, for example, A. N. Theofilopoulos and F. J. Dixon, Advances in Immunology, Volume 28, Pages 89-220 (1979) and S. E. Ritzmann and J. C. Daniels, Clinical Chemistry, Volume 28, Pages 1259-1271 (1982).
Because of this interest in detecting and monitoring the levels of circulating immune complexes, numerous tests have been described for detecting these complexes. These prior art tests have generally been based on: (a) the detection of epitopes contained in these complexes, (b) the reactivity of these complexes with radiolabeled reagents capable of binding to them, or (c) the binding of the immune complexes to specific receptors found on tissues or cells. An extensive body of art exists describing these tests.
These prior art tests have, however, required the isolation of the putative immune complexes to be detected from human serum because of the presence in the serum of other substances which would cross-react in the test and thus interfere with the detection of the immune complexes. This isolation of the immune complexes from serum has generally been accomplished in the prior art by precipitation (e.g., with polyethylene glycol), by gel filtration chromotography, or by solid phase columns.
The necessity in the prior art tests of isolating the circulating immune complexes from serum prior to performing the test is a difficult, expensive, and time-consuming step. A method which would eliminate the need for immune complex isolation from serum by allowing detection of immune complexes in serum would be of significant advantage. Such an assay would result in significant time saving in performing immune complex assays, would eliminate errors in prior art testing introduced during the isolation step (e.g., incomplete isolation or recovery of the immune complexes), and would eliminate errors introduced by the denaturation of the immune complexes during isolation.
Most antibodies present in immune complexes have a site on the Fc portion of the molecule which reacts with a component of complement. The complement system is part of a complex series of reaction by means of which materials having antibodies bound to them are destroyed by the host's immune system. These complement components are identified by letter and number abbreviations beginning with a capital C. One of particular interest is the first complement component (Cl), which itself is made up of subunits designated Clq, Clr, and Cls. It is the Clq subunit of Cl which reacts with the Fc site referred to above.
One of the prior art methods for detecting immune complexes has depended upon the detection of bound Clq (that is, Clq which is attached to the Fc site of antibody in the immune complex). The difficulty with accomplishing such a test in the presence of serum, however, is that prior art materials which have reacted with bound Clq have also reacted with Cl (which contains Clq) circulating in serum. Since this serum Cl is greatly in excess compared to the Clq bound to the complex, it has previously been impossible to conduct a Clq detecting assay in serum.
Since the seminal work of Kohler and Milstein in 1975, much effort has been directed to the production of various hybridomas producing so-called monoclonal antibody. These monoclonal antibodies are extremely useful materials because they have a single specificity, as opposed to so-called polyclonal antibodies resulting from the traditional immunization and bleeding of experimental animals.
Work in this area has simultaneously indicated the rewards and complications of attempting to produce monoclonal antibody from hybridomas. While the general technique is well understood conceptually, there are many difficulties met and variations required for each specific case. In fact, there is no assurance prior to attempting to prepare a given hybridoma that the desired hybridoma will be obtained, that it will produce antibody if obtained, or that the antibody so produced will have the desired specificity. The degree of success is influenced principally by the type of antigen employed and the selection technique used for isolating the desired hybridoma.
Recently, M. D. Golan and co-workers have used the technique of Kohler and Milstein to produce a variety of monoclonal antibodies to Clq and have used these antibodies to study conformational changes in Clq. They reported this work in the Journal of Immunology, Volume 129, Pages 445-447 (August 1982). These antibodies were produced by injecting mice with Clq bound to immune complexes as the antigen, fusing spleen cells from these mice with mouse myeloma cells, culturing the resulting hybridomas, and screening them against human Clq attached via rabbit anti-human Clq to a solid phase. These antibodies reacted with a variety of antigens, but all of them reacted with native serum Cl. Many of the antibodies also reacted with fluid-phase Clq. However, these antibodies generally reacted poorly with Clq bound to immune complexes.
The authors concluded that "[t]hese findings demonstrate that the binding of Clq to immune complexes exposes new antigenic determinants". They reached this conclusion because loss of reactivity with their antibodies resulted when the Clq bound to the immune complexes. The antibodies disclosed in this reference would not be useful for a test for immune complexes in serum by detection of Clq bound to immune complexes, because the native serum Cl would clearly obscure any result. In fact, this article appears to suggest that the monoclonal antibody technique would generally not be useful for specific detection of immune complexes.
We have now found that a specific test for immune complexes in serum can be devised using monoclonal antibodies directed at unique epitopes characteristic of Clq bound to immune complexes which are not displayed in native serum Cl.